Project Summary Proper axon outgrowth and pathfinding is essential for establishing precise neural networks of the nervous system during development. Defects in axon guidance are implicated in a variety of neurological disorders. Spatiotemporal regulation of guidance receptor expression facilitates differential responses of neurons to guidance cues in order to form accurate neuronal wiring. In vertebrates, levels of the guidance receptor Robo1 are low in precrossing and high in postcrossing commissural axons (CAs), functioning as a ?molecular switch? to regulate sensitivity to Slit repulsion and guide CA midline crossing. However, the mechanism underlying the fine-tuned spatiotemporal regulation of Robo1 expression remains largely unknown. MicroRNAs (miRNAs) regulate target gene expression by binding specifically to the 3?untranslated region (3?UTR) of target mRNAs, thus repressing translation and/or inducing mRNA degradation. Our preliminary studies indicate that chicken Robo1 (cRobo1) 3?UTR is required for regulation of protein expression in developing chicken spinal cords. miR-92, a highly conserved miRNA, suppresses cRobo1 expression in a miR-92 miRNA response element (MRE)-dependent manner. miR-92 and cRobo1 are differentially expressed in the developing chicken spinal cord with a mutually exclusive expression pattern. Ectopic expression of miR-92 in postcrossing commissural neurons results in CAs stalling in the floor plate. Therefore, we propose that miR-92 is a negative regulator of Robo1 expression in CAs by targeting its mRNA at the 3?UTR, thereby regulating Slit sensitivity to control CA projection and midline crossing. To test this hypothesis, we will first determine whether endogenous miR-92 specifically regulates cRobo1 expression in commissural neurons of embryonic chicken spinal cords during midline crossing (Aim 1): we will (1) examine the activity of endogenous miR-92 in precrossing commissural neurons of chicken spinal cords, (2) determine the subcellular expression patterns of miR-92 and cRobo1 in commissural neurons, and (3) untangle the mechanisms underlying miR-92-mediated repression of cRobo1. Secondly, we will focus on studying the functional importance of miR-92 in Slit/Robo1-mediated CA outgrowth and turning in vitro and CA projection and pathfinding in vivo (Aim 2). Finally, we will identify novel miRNAs targeting to cRobo1 in commissural neurons and examine their roles in Slit/Robo1-mediated CA guidance during midline crossing (Aim 3). These proposed experiments will support a model that specific miRNAs suppress Robo1 expression, thereby modulating Slit sensitivity to control Slit/Robo1- mediated CA guidance during embryonic spinal cord development.